The present invention generally relates to the individual stereoisomers of the drug valnoctamide (a mixture of four stereoisomer kinds, VCD-valmethamide or 2-ethyl-3-methyl pentanamide) useful in treatment of neurological and psychotic disorders such as different kinds of epilepsy and affective disorders, and useful as tranquilizers and to treat pain, and to pharmaceutical compositions containing, as an active ingredient, these stereoisomers. The present invention further relates to a method for stereoselective separation and quantification of the four stereoisomers from a racemic mixture of VCD or plasma of patients treated with the racemic drug.
The present invention further relates to a unique method for the synthesis of the individual stereoisomers.
Epilepsy is an ancient disease which affects about 1% of the global population. Despite the progress in antiepileptic therapy, about 25% of the epileptic patients continue to suffer from uncontrolled seizures and medication toxicity. At present, there are four major epileptic drugs in use: phenobarbital, phenytoin, carbamazepine and valproic acid. Valproic acid (VPA) is one of the major antiepileptic drugs. It has two major side effects, teratogenicity and hepatotoxicity, which have been associated with valproate therapy. Valnoctamide (VCD-valmethamide or 2-ethyl-3-methyl pentanamide) is an isomer of valpromide (VPD) and is sold as an over- the-counter drug in several European countries. It has been available clinically for many decades and is still used as a mild tranquilizer and occasionally as an antiepileptic drug (Chambon JP, Perio A, Neurosci. Lett [Suppl]5: S327-S327, 1980). Valpromide (VPD) is used as an anticonvulsant and antipsychotic agent, and in humans it is a prodrug of valproic acid (VPA). VPD may also be useful in treatment of neurological disorders and psychotic or affective disorders such as convulsions and epilepsy, and useful as tranquilizers and to treat pain. Recently, interest in VCD was revived by the observation that this compound possesses marked anticonvulsant activity in animal models. Both VCD and VPD are three times more potent as anticonvulsants than VPA. In addition, VPD, unlike VPA, has not been found to be teratogenic in animals possibly because it contains a carboxamide moiety instead of a carboxylic acid. However, in humans VPD acts as a prodrug to VPA and therefore its superiority over VPA in animal models does not have clinical implications. Unlike VPD, VCD acts as a drug on its own in both animals and humans and undergoes only slow and very little transformation to its corresponding (less active) valnoctic acid (VCA). Based on its anticonvulsant potency, metabolic stability and lack of teratogenicity, VCD was viewed as having potential to become a new antiepileptic drug. It was recently found, however, that VCD is an inhibitor of the enzyme epoxide hydrolase. This inhibition was regarded as a drawback to the development of racemic VCD as a new antiepileptic drug. In the present invention, for the first time, characterization of the pharmacokinetics (PK) of the four stereoisomers of VCD in humans was performed This characterization clearly demonstrates that VCD pharmacokinetics is stereoselective, with one isomer exhibiting a much higher clearance and a shorter half-life compared with the other stereoisomers. Stereoselective pharmacokinetics has been demonstrated previously with different drugs, such as verapamil and mephenytoin, but this is the first time that PK stereoselectivity has been shown for an amide of an aliphatic short-chain fatty acid.
The present invention relates to the stereoisomers of valnoctamide which are useful in treatment of neurological and psychotic disorders such as different kinds of epilepsy and affective disorders, and useful as tranquilizers and to treat pain. The present invention further relates to a method for stereoselective separation and quantification of the four stereoisomers from a racemic mixture of VCD and to a method for the synthesis of the 2S, 3S and 2R,3S VCD stereoisomers.
The stereoisomers have all of the benefits, and more, of the racemic drug of valnoctamide, but do not have any of its drawbacks, making them preferable for use in the treatment of convulsions and epilepsy and as an active ingredient in pharmaceutical compositions for the treatment of the latter. The present invention further relates to a method for separating the four stereoisomers of racemic valnoctamide. This method is a new stereoselective gas chromatography-mass spectrometry (GC-MS) assay which allows specific and sensitive quantitation of valnoctamide stereoisomers in human plasma. This method is the only one available for investigating the pharmacokinetics and pharmacodynamics of valnoctamide stereoisomers in plasma samples, investigations which may have important practical implications for the development of a new anticonvulsant drug for humans. The present invention further relates to a unique method for the synthesis of the individual stereoisomers.
The present invention relates to stereoisomers of valnoctamide (one of four isomers of valnoctamide) useful in treatment of neurological and psychotic disorders such as different kinds of epilepsy and affective disorders, and useful as tranquilizers and to treat pain, and to pharmaceutical compositions, containing as an active ingredient stereoisomers or a stereoisomer of valnoctamide, useful as anticonvulsant drugs or tranquilizers.
The present invention further relates to a method for separating valnoctamide isomers comprising subjecting a mixture of racemic valnoctamide to gas chromatography preferably with oven temperature programming conditions; 50xc2x0 C. for 1 minute, increasing at a rate of 8xc2x0 C./minute until 100xc2x0 C., holding for a minute then reaching final temperature of 250xc2x0 C. at a rate of 4xc2x0 C./minute; the injector temperature is 240xc2x0 C. and the GC-MS transfer line temperature is 250xc2x0 C.; inlet pressure is 5 psi with a linear flow rate 20 cm/second and the GC carrier gas is helium. Following, the isomers are separated on a chiral stationary phase column, preferably consisting of octakis (3-O-butanoyl-2,6-di-o-pentyl)-xcex3-cyclodextin chemically linked via a 6-mono-octamethylene spacer to a dimethylpolysiloxane and being coated on a fused silica capillary column of 25 mxc3x97250 xcexcm coated by 0.25 xcexcm octakis (3-O-butanoyl-2,6-di-o-pentyl)-xcex3-cyclodextrin.
The present invention further relates to a method of separating valnoctamide isomers comprising subjecting a mixture of racemic valnoctamide to gas chromatography preferably with oven temperature programming conditions; 50xc2x0 C. for 1 minute, increasing at a rate of 8xc2x0 C./minute until 100xc2x0 C., holding for a minute, then reaching final temperature of 250xc2x0 C. at a rate of 4xc2x0 C./minute; the injector temperature is 240xc2x0 C. and the GC-MS transfer line temperature is 250xc2x0 C.; inlet pressure is 5 psi with a linear flow rate of 20cm/sec and the GC carrier gas is helium. Following, the isomers are separated on a chiral stationary phase column, preferably consisting of octakis (3-O-butanoyl-2,6-di-o- pentyl)-xcex3-cyclodextrin chemically linked via a 6-mono-octamethylene spacer to a dimethyl polysiloxane and being coated with a fused silica capillary column of 25m X 250 xcexcm coated with 0.25 xcexcm octakis (3-O-butanoyl-2,6-di-o-pentyl)-xcex3-cyclodextrin.
The present invention relates to stereoisomers of the drug, valnoctamide (VCD-valmethamide or 2-ethyl-3-methyl pentanamide), useful in treatment of neurological and psychotic disorders such as different kinds of epilepsy and affective disorders, and useful as tranquilizers and to treat pain, and to pharmaceutical compositions containing, as an active ingredient, these stereoisomers. VCD, as compared with the well-known antiepileptic drug VPA, is retained in the body for a relatively long period and the plasma levels of its corresponding acid (VCA) are much lower than those of the parent drug. In contrast to VPD, which has a high (metabolic) extraction ratio by the liver, where it undergoes extensive first-pass metabolism to its corresponding acid VPA, VCD does not have a high affinity for hepatic metabolism. This is reflected by its low oral clearance and its half-life, which is ten times higher than that of VPD (Bialer et al., Eur. J. Clin. Pharmacol. 38: 289-291, 1990). Based on its anticonvulsant potency, metabolic stability and lack of teratogenicity. VCD was viewed as having potential to become a new antiepileptic drug. However, it was recently found (Pisani et al., Epilepsia 34:954-959, 1993) that VCD is an inhibitor of the enzyme epoxide hydrolase. The inhibition of this enzyme, resulting in the decreased clearance of CBZ-E in patients who were treated with the antiepileptic drug carbamazepine (CBZ) together with VCD, caused in these patients signs suggestive of CBZ intoxication. This inhibition was regarded as a drawback to the development of racemic VCD as a new antiepileptic drug, but since VCD has two chiral centers (as depicted in FIG. 1), and there are two pairs of enantiomers in the VCD molecule, there is, a possibility that its pharmacokinetics (PK) and pharmacodynamics (PD), including toxicological potential and epoxide hydrolase inhibition, are stereoselective. By itself, PK stereoselectivity could lead to PD stereoselectivity, and have considerable implications for the further development of an antiepileptic drug. The method for the stereoselective separation and quantfication of the four stereoisomers from a racemic mixture of VCD or plasma of patients treated with the racemic drug, of the present invention was developed based on this background. This method is a new stereoselective gas chromatography-mass spectrometry (GC-MS) assay which allows the specific and sensitive quantitation of VCD stereoisomers.
The present invention further relates to a method for the stereoselective synthesis of the valnoctamide stereoisomers (2S,3S) and (2R,3S). The said method of synthesis comprises the following steps;
(a) synthesizing (3S)-methyl valeric acid (see FIG. 6(I)) from L-isoleucine;
(b) synthesizing of (3S)-methyl valeroyl chloride (see FIG. 7(II));
(c) synthesizing (2S,3S) valnoctic acid (see FIG. 8(IIIxe2x80x2)) or subsequently synthesizing (2R,3S) valnoctic acid (see FIG. 8(III));
(d) synthesizing (2S,3S) valnoctamide (see FIG. 8(Vxe2x80x2)) or (2R,3S) valnoctamide (see FIG. 8(V)). The said invention will be further illustrated by the following experiments. These experiments do not intend to limit the scope of the invention, but to demonstrate and clarify it only. The method of separation of the present invention was applied to investigate the pharmacokinetics of VCD stereoisomers in plasma samples from seven healthy subjects and six epileptic patients.
Healthy Subjects Seven healthy male volunteers, aged 20-31 years, weighing 61 to 85 kg, received 2xc3x97200 mg valnoctamide tablets (Nirvanil@) at 08:00 hours after an overnight fast. Venous blood samples were taken through an indwelling catheter 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12 and 24 hours after dosing. Food was witheld for 4 hours after dosing.
Patients Six epileptic patients (four men and two women aged 23-35 years, with a body weight of 56-85 kg) were studied. All had been receiving a constant carbamazepine dose (800-1200 mg/day) for at least three months. One patient was also receiving phenytoin (250 mg/day) and a second patient phenobarbital (150 mg/day). Each patient received add-on VCD for eight consecutive days at a dosage of 400 mg on day 1 (single dose day), followed by 200 mg three times daily (tid) for seven days. On the last day, only the morning and the midday doses were administered. Blood samples for plasma VCD levels were collected at 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12 and 24 hours after the first and the last morning dose. Additional samples were taken at times 0, 0.5, 1, and 2 hours after the morning dose on the last treatment day.
Plasma Handling and Analytical Assays Plasma samples were separated immediately by centrifugation at 2,000 g for 10 minutes and stored at xe2x88x9220xc2x0 C. until assaying (VCD is stable at xe2x88x9220xc2x0 C.). The VCD plasma samples were analyzed by a gas chromatograph (HP 5890 series II)-mass spectrometer (HP 5989A)-GC-MS data system equipped with chiral capillary column. The mass spectrometer was operated in E1 mode at 70eV, the source temperature of the MS was maintained at 200xc2x0 C. and the quadruple at 100xc2x0 C. Mass units and their relative abundance were calibrated with perfluorotributylamine (PFTBA). Spectra were collected in the range of 35 to 350 MHz, and the chiral analysis started 18 minutes after injection. Gas chromatograph (GC) oven temperature programming conditions were as follows: 50xc2x0 C. for 1 minute, then increasing at a rate of 8xc2x0 C./minute until 100xc2x0 C., holding for a minute then 4xc2x0 C./minute until reaching a final temperature of 250xc2x0 C. The injector temperature is 240xc2x0 C. and the GC-MS transfer line temperature is 250xc2x0 C. Inlet pressure is 5 psi with a linear flow rate 20 cm/second. Helium was used as the carrier gas. Chromatographic resolution of the VCD stereoisomers was accomplished by a direct separation on a chiral stationary phase (CSP) column. The CSP consisted of a Chirasil-xcex3-Dex (octakis (3-O-butanoyl-2,6-di-o-pentyl)-xcex3-cyclodextrin chemically linked via a 6-mono-octamethylene spacer to a dimethylpolysiloxane. The 0.25 xcexcm Chirasil-xcex3-Dex was coated on a 25 mxc3x97250 xcexcm (id) fused silica capillary column. This CSP of a cyclodextin derivative allows complete baseline resolution of the four stereoisomers (xcex1=1.04, Rs=2.0-2.4) in 24 minutes as depicted in FIG. 2. Intra and inter-day variability in the retention times of the four VCD stereoisomers was less than 10%. A calibration curve was established by spiking drug-free plasma samples with known amounts of racemic VCD at a concentration range of 0.2 mg/L to 8 mg/L (or 0.05 mg/L to 2 mg/L of each of the four VCD stereoisomers).
Reagents and Materials VCD (racemic mixture) was obtained from Clin Midy (Italy). Propylisopropylacetamide (PID), previously synthesized by Haj-Yehia and Bialer (Pharm Res 6:683-9,1989), was used as internal standard (IS). Stock solution of VCD (racemic mixture) was prepared by dissolving the drug in chloroform at a concentration of 5 mg/mL. The internal standard (PID) was dissolved in chloroform at a concentration of 10 mg/mL. The stock solutions were stored at xe2x88x9220xc2x0 C.
Pharmacokinetic and Statistical Analysis Pharmacokinetic parameters were calculated by noncompartmental analysis according to statistical moment theory. Peak plasma concentrations (Cmax) and peak times (tmax) were derived directly from the measured values. The linear terminal slopes (xcex) of the log-concentration of each VCD stereoisomer versus time curves were calculated by the peeling procedure. Half life (txc2xd) values were calculated as 0.693/xcex. Areas under the plasma concentration versus time curve (AUC), after single dosing, were calculated by the trapezoidal rule with extrapolation to infinity by dividing the last measured concentration by the terminal slope. During repeated administration AUC was calculated over the last two dosing intervals by using the trapezoidal rule. Mean residence time (MRT) was calculated after single dosing as AUMC/AUC, in which AUMC is the area under the concentrationxc3x97time product versus time curve from zero to infinity. Following single dosing, oral clearance (CL/F, in which F is the oral availability) and apparent volume of distribution at steady state (Vss/F) were calculated as Dose/AUC and as MRTxc3x97CL/F, respectively. Following repeated dosing, CL/F was calculated from the quotient of the dose (200 mg) and the AUC obtained during one dosing interval (defined as one-half of the AUC calculated over two consecutive dosing intervals). Comparison of the pharmacokinetic parameters of all four VCD stereoisomers was made by ANOVA.
Results The stereoisomers of VCD were identified as A, B, C and D based on their respective retention times as shown in FIG. 1. As a step towards characterization, enantiomeric pairs of diastereisomers were separated by repeated crystallization of racemic VCD. After eight re crystallizations, a fraction of one pair of enantiomers (A and C) contained only 12% of the other enantiomeric pair (B and D). Intra-day and inter-day coefficients of variation for each analyte were below 10%. FIG. 2 shows a typical GC-MS chromatogram depicting the high resolution of the four stereoisomers (peaks A, B, C, and D) of VCD. The mass spectrum of each of the four VCD stereoisomers was identical. The mean plasma concentrations of the four stereoisomers following single oral dosing of racemic VCD to healthy subjects and epileptic patients are shown in FIGS. 3 and 4, while FIG. 5 depicts the mean plasma concentrations of the stereoisomers during the last day after eight days of repeated oral dosing of racemic VCD in epileptic patients. FIG. 5 exhibits primary and secondary peaks due to the fact that on the last day only 2 hours elapsed between the morning and the midday doses. The individual and mean pharmacokinetic parameters of the stereoisomers in healthy subjects and epileptic patients are given in Tables 1 to 3. In healthy subjects stereoisomer B had the largest oral clearance (CL/F=8.7+xe2x80x940.9 L/h) and its half-life (txc2xd) and mean residence time (MRT) were the shortest of all VCD stereoisomers. Following single doses of VCD in epileptic patients, stereoisomer B also showed the largest oral clearance (80+xe2x80x9420L/h) and the largest volume of distribution (Vss/F=349+xe2x80x9460 L) compared to all the other stereoisomers. For all stereoisomers, CL/F values in epileptic patients were about 10-fold greater than those determined in healthy subjects who received the same dose under identical conditions. Vss/F values were also much larger in patients than in healthy subjects. Because of their short half-lives, all stereoisomers showed negligible accumulation during repeated dosing (FIG. 5 and Table 3). After 7-day dosing, however, CL/F values at steady-state were lower than those determined in the same patients after a single dose.
This study provides the first PK characterization of the four stereoisomers of VCD in man. When the concentration of individual isomers in plasma was added up at each sampling time, the resulting values were in close agreement with those determined previously in the same samples by using a different non-stereoselective GC assay (Pisani et al., Epilepsia 34:954-959, 1993). This provides confirmatory evidence for the reliability of the assay used in this study and for the stability of VCD under the indicated storage conditions. Evaluation of the kinetic properties of individual isomers in healthy subjects and in epileptic patients receiving concomitant anticonvulsant therapy clearly demonstrated that VCD pharmacokinetics is stereoselective, with one isomer (stereoisomer B) exhibiting a much higher clearance and a shorter half-life compared to other isomers. In fact, a pronounced difference in PK behavior was observed not only between stereoisomer B and its diastereoisomers A and C, but also between individual enantiomers B and D. Previous studies have shown that VCD is completely absorbed and is eliminated predominantly by metabolism. Therefore, the difference in PK properties between individual stereoisomers must involve a stereoselective process in the metabolism of the drug. In this respect, the larger apparent volume of distribution (Vss/F) of stereoisomers B, which was most clearly evident in epileptic patients, could be related to higher first -pass metabolism of this isomer, resulting in reduced oral bioavailability. Compared with healthy control subjects, epileptic patients exhibited an approximately 10-fold greater apparent oral clearance (CL/F) for each of the four stereoisomers. Epileptic patients also showed shorter half lives compared with control (about 3 h vs. 10 h for stereoisomers A,C and D, and about 3 h vs. 5.8 h for stereoisomer B). The promiment VCD elimination between healthy subjects and epileptic patients could be explained by induction of the oxidative metabolism of VCD stereoisomers by carbamazepine which is a potent inducer of cytochrome P450. Indeed the minimal urinary excretion of unchanged VCD and its minor metabolic hydrolysis to its corresponding acid VCA suggest that oxidative metabolic pathways play an important role in VCD elimination. Induction of first-pass metabolism by carbamazepine may also explain the larger volumes of distribution (Vss/F) of VCD stereoisomers in patients with epilepsy. The decrease in apparent oral clearance of VCD isomers which was observed during repeated dosing in epileptic patients, on the other hand, is suggestive of time-dependent changes in VCD disposition, possibly related to concentration-dependent metabolism of the drug. Although stereoselective pharmacodynamics is not necessarily associated with PK stereoselectivity, the differences in PK behavior of VCD stereoisomers increase the likelihood that differences also exist in their anticonvulsant activity or undesired properties such as teratogenicity, hepatotoxicity and epoxide hydrolase inhibition. A unique method for the synthesis of individual stereoisomer was developed and their absolute configuration was resolved by X-RAY diffraction analysis as shown in FIG. 9.